Interest in the Rho family of small GTPases has dramatically intensified in the last few years. Although a critical role for these proteins in regulating the actin cytoskeleton has been revealed, the organization and in vivo function of Rho-associated signaling pathways is still largely unclear. Accumulating evidence has revealed that the Rho GTPases mediate the various morphogenetic events associated with embryogenesis, and recently, these proteins have also been implicated in tumorigenesis. To advance the understanding of Rho proteins, studies have been initiated of closely related Rho homologs in Drosophila, a powerful genetic system. These studies have led to several findings: 1. Drosophila Rho GTPases, when overexpressed in the fly eye, disrupt the normal developmental program, giving rise to a "rough eye". 2. In a screen to identify suppressors of the Rho1-induced rough eye, several Rho1-specific mutant modifier alleles were identified. 3. Several of these alleles were found to correspond to a novel Rho-specific guanine nucleotide exchange factor, DRhoGEF2. 4. DRhoGEF2 is required for the cell shape changes in gastrulation and mediates a Rho signaling pathway in response to the extracellular ligand, Fog. In addition to DRhoGEF2, a Drosophila homolog of the major mammalian Rho effector target kinase, PKN, was also identified. It was determined that PKN, together with Rho, is required for the cell shape changes during the developmental process of dorsal closure. In short, Rho-mediated signaling pathways have been identified that are required for the two major morphogenetic events of Drosophila embryogenesis-- namely, gastrulation and dorsal closure. These observations will serve as the basis for further studies aimed at establishing the precise role of these identified Rho pathway components in the regulation of tissue morphogenesis. This will be accomplished through a detailed phenotypic analysis of loss-of-function alleles of the identified genes, genetic interaction studies with existing signaling pathway mutants, and biochemical studies aimed at determining the molecular organization of the identified Rho-mediated pathways. In addition, studies are proposed to address the mechanism by which distinct GTPase-mediated pathways can function to coordinately regulate a single developmental process. The long-term goal of these studies is to advance the current understanding of the normal biological function of Rho GTPases and the organization of their associated signaling pathways, with particular emphasis on establishing their role in tissue morphogenesis.